Nucleic acid molecules encoding proteins which impart the adhesion of Neisseria cells to human cells

ABSTRACT

Described are nucleic acid molecules encoding proteins mediating the adhesion of bacteria of the genus  Neisseria  to human cells. Also described are the proteins encoded by these nucleic acid molecules and antibodies directed against them. Furthermore, pharmaceutical compositions, vaccines and diagnostic compositions containing the nucleic acid molecules, proteins and/or antibodies are described.

The present invention relates to nucleic acid molecules from bacteria ofthe genus Neisseria encoding proteins mediating the adhesion ofNeisseria cells to human cells. Furthermore, the present inventionrelates to the proteins encoded by these nucleic acid molecules and toantibodies directed against them. The present invention further relatesto pharmaceutical compositions, vaccines and diagnostic compositionscontaining said nucleic acid molecules, proteins and/or antibodies.

To the genus Neisseria (gram-negative cocci) belong a number ofbacterial species which, being saprophytes, populate the upper humanrespiratory tract. Apart from commensal species (e.g.: N. sicca) andopportunistically pathogenic species (e.g.: N. lactamica), two Neisseriaspecies are known which clearly possess human-pathogenic properties. Oneof the species is N. gonorrhoeae, the pathogen of the venereal diseasegonorrhea, which exclusively occurs in humans, and N. meningitidis, thepathogen of the bacterial epidemic meningitis. In both cases theetiology, that is the causal connection between the development of theclinical picture and the population by bacteria from said species hasmeanwhile been substantiated.

The purulent meningitis (Meningitidis cerebrospinalis epidemica) causedby N. meningitidis (“meningococcus”), which usually is epidemical, is asystemic invasive infection of the human meninx and spinal meninx.Occasionally, hemorrhagic exanthema at the trunk or concomitant diseasescaused by Herpes simplex can be observed in addition. The pathogen canappear in the form of several serotypes, which are distinguishable bymeans of agglutination assays with immune sera. The main groups differremarkably, and their prevalence differs with regard to when and wherethey appear. Meningococcus meningitidis has up to now occurred in largenumbers every 8 to 12 years with the increased prevalence lastingbetween 4 to 6 years. While serovar B meningococci brought on 50% to 55%of the recent diseases to the civilian population as well as to themilitary personnel in the United States, most epidemic diseases in theUnited States during the first half of the century were caused byserovar A meningococci.

The clinical picture caused by N. gonorrhoeae usually is an infectionlocalized to the mucous membranes, in most cases of the urogenital tract(gonorrhea), more rarely of the conjunctiva (conjunctivis gonorrhoeae,gonoblennorrhoe), which is acquired by new born children perinatally, byadults usually unilaterally by smear infection. In very rare casesbacteremia and sepsis occur after hematogenous dissemination. As aconsequence, exanthema with hemorrhagic pustules, diseases from therheumatic Formenkreis, arthritis gonorrhoica and/or endocarditis canoccur.

Usually, the diseases caused by N. gonorrhoeae and N. meningitidis aretreated with antibiotics. More and more, however, the bacteria arebecoming resistant to single or groups of the antibiotics used so thatthe therapy method that has nearly exclusively been used up to now willmost likely not be successful in the long run. Therefore, it isdesirable and urgent that alternative therapy methods, preferablypreventive ones, be developed.

Neisseria gonorrhoeae and N. meningitidis exclusively occur in humans.They have adapted to the host organism and show a number of propertiesthat are able to make the defense mechanisms of the host ineffective.Therefore, up to now there is no vaccine available that preventsgonorrhea. This is to a limited extent also true for meningococcusmeningitidis. Even though the disease has recently been caused mainly bybacteria of the same serovar, group B, no effective vaccine againstmeningococci of group B has existed up to now. Vaccines against otherserovars only offer partial protection and are not unproblematic from animmunological point of view. The reason for the failure of the immunedefense is, inter alia, the antigen variation of the pathogens, which inthe case of the pathogenic Neisseria is particularly developed. However,a limitation of the free development of the antigen variation seems tobe necessary where the functional region has to be sterically maintainedin order to guarantee the interaction with conserved and constantstructures of the host receptors. This requirement especially applies tothe adhesins that serve for adhering to the host cell. Only if thefunctional region that is involved in the physical interaction is keptconstant, the interaction with the receptor of the host cell ispossible. This region should be excluded from antigen variation to alarge extent and is therefore a suitable starting-point for thedevelopment of a new therapy method.

The initial phase of infections usually is the stable adhesion of thepathogens to the host tissue. By interactions between structures of thecell surface of the pathogens and the cell surface of the host cell amechanically stable linkage is formed that allows the bacteria to stayon the tissue of the host (colonization) and to subsequently propagatelocally. The adhesion to the host cell can be divided into two phaseswith different structures being involved in the interaction.

In the first phase of adhesion a contact between host cell and pathogenis mediated. Often cell appendage organelles, the so-called pili, areinvolved in mediating the contact. These cell organelles, which are alsocalled fimbriae or fibrils, are few to several fine filamentous rigid orflexible appendages of the bacterial cell, which can be several times aslong as the cell diameter. Therefore, there is no contact between thecell walls of pathogen and host cell in the pilus mediated adhesion. Themajority of the known pili are heteropolymeric structures consisting ofseveral components. The main subunit, which usually is present in manycopies, fulfills the structural function, that is the frameworkfunction, whereas the actual adhesion function is fulfilled by sidecomponents, which usually are present in few copies.

A further form of adherence is the adhesion of pathogens to the hostcells without the contribution of pili (pilus independent adherence,pia). In this case, the pathogen and the host cell are approaching eachother, and finally the cell walls directly touch. This adhesion andstabilization of the contact between the cells takes place with thecontribution of adhesines that are located in the bacterial cell wall.As a result of the direct contact between the cells, a signal is finallytransmitted that initiates the pathogen induced phagocytosis and startsthe invasion process into the target cell. The pia form of adherence canautonomically effect the adhesion of pathogens, for example in the caseof pathogens lacking pili. It can, however, also act as the second phaseof adhesion, that is as the consecutive reaction after pilus mediatedadhesion, and stabilize the contact between the cells. The adhesinesthat are involved in the pilus independent adhesion can but do notnecessarily have to show different binding specifities from those thatare involved in pilus dependent adhesion.

In the context of the invention the bacterial structures that areinvolved in the adhesion will in the following be called adhesines,those of the host cells will be called receptors. If there is no contactbetween adhesin and receptor, “defense mechanisms” of the host, such asfibrillation of the epithelia, mucus secretion, mass flow of body fluidsand the like, eliminate the pathogens. The development of an infectionis, therefore, prevented from the very beginning. Thus, a disturbance ofthe adhesion of the pathogens by means of inhibiting the interactionbetween adhesin and receptor of the target cell represents a veryeffective approach for preventing and treating infections. Suchtherapeutically effective approaches comprise the production ofantibodies specifically blocking the adhesin function, either by activeimmunization (vaccination) or by administration of antibodies alreadyexisting (passive immunization). The adhesin receptor binding can, inthe same way, be inhibited by means of passive administration of bothreceptor analogous and adhesin analogous substances. These substancescompetitively bind to the corresponding partner structures, therebyblocking their involvement in productive interactions. In the context ofthe invention such substances are called inhibitors.

The approaches using pilin, the main component of the pilus thatfulfills the structural function, in order to develop a broadlyeffective vaccine effectively blocking the adhesion of pathogenicNeisseria have failed so far. The reason probably is that (i) pilinitself has no adhesin function and (ii) pilin possesses an especiallydistinct intra- and interstem specific antigenic variation. Since bothlimitations, as described above, do not apply to adhesins, the use of anadhesin as a vaccine is more promising.

The technical problem of the present invention therefore is to provideproteins and DNA molecules encoding them that serve as adhesionstructures for Neisseria species or contribute to the development ofsuch structures.

This problem is solved by providing the embodiments described in theclaims.

Therefore, the present invention relates to nucleic acid moleculescontaining the nucleotide sequence described in Seq ID No. 1 or partsthereof with these nucleic acid molecules comprising one or more openreading frames encoding proteins or biologically active fragmentsthereof from bacteria of the genus Neisseria that mediate the adhesionof Neisseria cells to human cells. The term “reading frame” in thiscontext is used synonymously with the term “coding region”.

The subject matter of the invention also relates to nucleic acidmolecules that basically show the nucleotide sequence described in SeqID No. 1 but whereby the nucleotide sequences of the open reading framesdeviate from those described in Seq ID No. 1 due to the degeneration ofthe genetic code. Preferably, the open reading frames of those nucleicacid molecules have nucleotide sequences encoding proteins with one ofthe amino acid sequences described in Seq ID No. 1. The subject matterof the invention further relates to nucleic acid molecules hybridizingto the nucleic acid molecules described above and comprising codingregions encoding proteins that mediate the adhesion of Neisseria cellsto human cells.

In the context of the present invention the term “hybridization” is usedas described in Sambrook et al. (Molecular Cloning, A Laboratory Manual;Cold Spring Harbor Laboratory Press (1989), 1.101 to 1.104). Preferably,this term has the meaning of hybridization under stringent conditions.In particular, it has the meaning of a hybridization that still shows apositive hybridization signal after being washed for 1 h with 1×SSC and0.1% SDS, preferably with 0.2×SSC and 0.1% SDS, at 55° C., preferably at62° C. and most preferably at 68° C.

In a preferred embodiment the nucleic acid molecule of the inventionoriginates from a pathogenic Neisseria species, in particular fromNeisseria gonorrhoea or Neisseria meningitidis.

The term “nucleic acid molecule” as used here according to the inventionrelates to the polymeric form of nucleotides of any length, either asribonucleotides or as desoxyribonucleotides. The term only relates tothe primary structure of the molecule. In this sense, it comprises DNAand RNA molecules, in single- or double-stranded form. The DNA caneither be cDNA or genomic DNA. The term further comprises thenon-modified form as well as scientifically known modifications, e.g.,methylation, capping, base substitution with natural or syntheticanalogues, internucleotide modifications with uncharged compounds (e.g.,methyl phosphate, phosphoamidate, carbamate, phosphotriester and thelike) or with charged compounds (e.g., phosphorothioate,phosphorodithioate and the like) or with binding components such asproteins and peptides (e.g., nucleases, toxins, antibodies,poly-L-lysine, and the like). The term also comprises forms withintercalating substances (e.g., acridin, psoralen, and the like),chelators (e.g., with metals, radioactive metals or oxidizing metals andthe like), with alkylating agents and finally with modified bonds (e.g.,alpha anomeric nucleic acids, and the like).

The invention also relates to vectors containing a nucleic acid moleculeof the invention. The vector can be any prokaryotic or eukaryoticvector. Examples of prokaryotic vectors are chromosomal vectors, such asbacteriophages (e.g., bacteriophage lambda, P1), and extrachromosomalvectors, such as plasmids with circular plasmids being particularlypreferred. Suitable prokaryotic vectors are, for example, described inSambrook et al. (see above), chapters 1 to 4. The vector according tothe invention can also be a eukaryotic vector, for example a yeastvector or a vector suitable for higher cells (e.g., a plasmid vector, aviral vector, a plant vector, and the like). Examples of such vectorsare also described in Sambrook et al. (see above, chapter 16). A vectorcontaining a nucleic acid molecule of the invention is, for example,plasmid pES25 (contained in the E. coli strain H 2560 (DSM 10257)). TheE. coli strain H 2560 was deposited on Sep. 18, 1995 with DeutscheSammlung von Mikroorganismen (DSM) [German collection of microorganisms]in Brunswick, Federal Republic of Germany, as international recognizeddepositary authority in accordance with the stipulations of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure under accession number DSM 10257.

The invention furthermore relates to host cells containing a vector asdescribed above or being genetically manipulated with a nucleic acidmolecule as described above. The term “host cell” in the context of thisinvention comprises both prokaryotic and eukaryotic host cells.Prokaryotic cells are preferred, particularly gram-negative prokaryoticcells, in particular E. coli cells. Suitable eukaryotic host cells are,for example, fungal cells (e.g., yeast cells), animal or plant cells.

The nucleotide sequence described in Seq ID No. 1 comprises three openreading frames. They represent an operon forming a functional unity. Thethree open reading frames called orfl, orfA and orfB encode threeproteins that in the context of this invention are called OrfI, OrfA andOrfB. These sequences are responsible for the expression of a protein inNeisseria cells, in particular of the protein OrfA, which is involved inthe adhesion of Neisseria cells to human cells. The proteins OrfI andOrfB obviously possess a regulatory function or a function as factorsthat are able to influence the functionality of OrfA.

This nucleic acid molecule therefore represents a region of theNeisseria genome that encodes proteins having the adhesin function ofNeisseria cells.

The present invention further relates to nucleic acid molecules encodinga lipoprotein or biologically active fragments thereof from bacteria ofthe genus Neisseria having the amino acid sequence as described in SeqID No. 2. In a preferred embodiment the invention relates to nucleicacid molecules encoding a protein having the amino acid sequence fromthe amino acid residue 19 to the amino acid residue 320 of the aminoacid sequence as described in Seq ID No. 2. Such nucleic acid moleculespreferably have the nucleotide sequence described in Seq ID No. 2, inparticular the nucleotide sequence from nucleotide 189 to nucleotide1095 of the sequence described in Seq ID No. 2.

The subject matter of the invention also relates to nucleic acidmolecules encoding a lipoprotein from bacteria of the genus Neisseriawhereby their nucleotide sequence deviates from the nucleic acidmolecules described above due to the degeneration of the genetic code.

Furthermore, the present invention relates to nucleic acid moleculesencoding a lipoprotein from bacteria of the genus Neisseria andhybridize to one of the nucleic acid molecules described above (for thedefinition of the term “hybridization” see above).

The subject matter of the invention also relates to fragments,derivatives and allelic variants of the nucleic acid molecules describedabove that encode the lipoprotein described above. Fragments areunderstood to be parts of the nucleic acid molecules that are longenough to encode the protein described. The term derivative in thiscontext means that the nucleotide sequences of these molecules differ atone or more positions from the sequences of the nucleic acid moleculesdescribed above and that they show a high level of homology to thesenucleotide sequences. Homology means a sequence identity of at least40%, in particular an identity of at least 60%, preferably of more than80% and particularly preferred of more than 90%. The deviations to thenucleic acid molecules described above can be caused by deletion,substitution, insertion or recombination.

Homology further means that there is a functional and/or structuralequivalence between the corresponding nucleic acid molecules or theproteins encoded by them. The nucleic acid molecules that are homologousto those described above and that represent derivatives of these nucleicacid molecules usually are variants of these molecules displayingmodifications that have the same biological function. They can benaturally occurring variants, for example sequences from otherorganisms, or mutations, which either occur naturally or that have beenintroduced by means of specific mutagenesis. Furthermore, the variantscan be synthetically produced sequences.

The allelic variants can be both naturally occurring variants orvariants that were synthetically produced or that were produced byrecombinant DNA techniques. The proteins encoded by the various variantsof the nucleic acid molecules according to the invention show certaincommon characteristics, for example enzyme activity, molecular weight,immunological reactivity, conformation etc., as well as physicalproperties such as the electorphoretic mobility, chromatographicbehavior, sedimentation coefficients, solubility, spectroscopicproperties, stability, pH optimum, temperature optimum etc.

Preferably, the proteins encoded by the nucleic acid molecules accordingto the invention show a homology of 80%, particularly preferred of morethan 90% to the nucleotide sequence described in Seq ID No. 2.

The nucleic acid molecules described above encode a lipoprotein frombacteria of the genus Neisseria. This protein is called OrfA in thecontext of the present invention. This protein is, according toexperimental data, located on the cell surface of Neisseria cells, inparticular on the outer membrane. The protein preferably has a molecularweight of about 36 kd if it is analyzed in the T7 expression system.

Furthermore, this protein possesses a biological activity that mediatesthe adhesion of Neisseria cells to human cells. This is in particularlymade because this protein forms a complex with the protein fromNeisseria known as PilC. The adhesion preferably takes place on humanepithelial cells.

Furthermore, the invention relates to vectors containing nucleic acidmolecules described above. Examples of such vectors have already beendescribed above.

In a preferred embodiment the DNA molecules according to the inventionare linked in such vectors with regulatory DNA elements that make theexpression of the protein in prokaryotic or eukaryotic cells possible.Examples thereof are in the context of this invention promoters,operators, enhancers and the like.

Furthermore, the invention relates to host cells that contain vectorsaccording to the invention described above or that have been geneticallymanipulated with the nucleic acid molecules described above. Geneticallymanipulated means that such a molecule has been introduced into the hostcell or in a precursor cell by means of (gene) technological methods.Again, the above-described host cells are suitable.

The invention also relates to methods for the production of thedescribed lipoprotein or a biologically active fragment thereof wherebythe host cells described above are cultivated under conditions thatallow the expression of the protein and the protein is isolated from thecells and/or the culture supernatant.

The invention also relates to proteins encoded by one of the nucleicacid molecules described above, as well as to biologically activefragments thereof as well as to proteins available by the methoddescribed above. In particular, the invention relates to proteins havingamino acid sequences that immunologically cross-react with the describedproteins. The term “protein” comprises in the context of the presentinvention also naturally occurring variants or modifications orfragments or synthetically produced modifications, variants or fragmentswith the corresponding biological activity. Derived or recombinantproteins do not necessarily have to be biologically translated from thenucleotide sequence. They can be produced in any way, including chemicalsynthesis, in vitro synthesis by means of an expression system or byisolation from organisms. Proteins according to the invention cancontain one or more amino acid analogues or amino acids not naturallyoccurring. Also, modifications (e.g., glycosylation, and the like) orlabeling (e.g., biotinylation) according to the scientific knowledge canbe contained.

The fragments preferably have a length of at least 3 to 5 amino acids,particularly preferred of 8 to 10 amino acids and in particularpreferred of 11 to 15 amino acids. This is also true for the proteinsaccording to the invention described below.

The lipoprotein OrfA according to the invention can be purified, forexample, by a method that is based on the interaction of this proteinwith the PilC protein from Neisseria gonorrhoeae. It is preferablypurified from homogenates of cells expressing this protein by means ofchromatography matrices containing immobilized PilC protein. The proteincan then be selectively eluted using its affinity to PilC and producedin essentially pure form.

The proteins according to the invention or fragments thereof can be usedas immunogens for the production of antibodies. Therefore, the presentinvention also relates to antibodies that are directed against a proteinaccording to the invention or a fragment thereof. The antibodies can beboth polyclonal and monoclonal. Methods for the production of suchantibodies are known to the skilled person.

In a preferred embodiment such antibodies are directed against epitopesof the protein according to the invention or fragments thereof that areimportant for the adherence and for the interaction with PilC. Theantibodies according to the invention can be, for example, produced byintroducing the nucleic acid sequences according to the inventiondescribed above into hosts by in vivo transfection. Thereby, the proteinor a fragment thereof is expressed in the host and the antibodiesdirected against them are induced (nucleic acid vaccination). This isalso the case with the antibodies described below.

The present invention further relates to nucleic acid molecules having alength of at least 12 nucleotides and specifically hybridizing to thenucleic acid molecule described above. Preferably, such nucleic acidmolecules have a length of at least 15 nucleotides, particularlypreferably of 20 nucleotides. Such molecules are, for example, suitableas primers for in vitro amplification, for example by polymerase chainreaction (PCR), or suitable for diagnostic purposes, that is forspecifically identifying the nucleic acid molecules of the invention insamples.

The invention further relates to pharmaceutical compositions containinga nucleic acid molecule according to the invention described above, aprotein, a biologically active fragment thereof and/or an antibodyaccording to the invention described above. In the context of thepresent invention such pharmaceutical compositions can contain the usualpharmaceutical adjuvants, diluents, additives and/or carriers. Theinvention also relates to vaccines containing the nucleic acid moleculesdescribed above, proteins, biologically active fragments thereof and/orantibodies.

In a further aspect the present invention relates to diagnosticcompositions containing the nucleic acid molecules according to theinvention described above, proteins, biologically active fragmentsthereof and/or antibodies.

A further aspect of the present invention relates to receptors andsubstances having receptor function, interacting as ligands with theadhesin according to the invention, the OrfA-PilC complex. Suchsubstances can be identified as competitive inhibitors of the adherencefunction due to their interaction with the OrfA-PilC complex. They canbe surface components of human cells, particularly preferred surfacecomponents of human epithelial cells or chemical substances of anyorigin.

Finally, the present invention relates to inhibitors that influence theinteraction between the OrfA-PilC adhesin complex and its receptors.Enclosed are all substances according to the invention that influencethe interaction between the OrfA-PilC adhesin and its cellular receptorand therefore disturb the adherence. In a particularly preferredembodiment substances that irreversibly bind to the adhesin complex suchas receptor analogues are encompassed.

Finally, the present invention relates to pharmaceutical compositionscontaining as an agent

-   (a) a receptor according to the invention;-   (b) a receptor analogue according to the invention; and/or-   (c) an inhibitor according to the invention,    optionally together with the usual pharmaceutical adjuvants,    diluents, additives and carriers.

The pharmaceutical compositions described in the context of the presentinvention can be used for identifying and characterizing a bacterialsample not yet known as pathogenic Neisseria spc. and for diagnosing aNeisseria infection.

On the polynucleotide level, preferably hybridization probes are usedcontaining the nucleotide sequences of the invention that are specificfor one of the orf-gene regions or nucleotide sequences of the inventionfrom one of the orf gene regions are used as primers for the PCRamplification of the genomic DNA region to be identified that isspecific for pathogenic Neisseria.

On the polypeptide level diagnosis is preferably performed with the helpof antibodies of the invention or, in the case of antibody screeningtests, with the help of immunogenic proteins of the invention orfragments thereof.

Receptors, receptor analogous substances and inhibitors of theinteraction between the OrfA of the invention and the correspondingreceptors of the host cells can be used as therapeutics for infectionsat an early stage or if an infection is suspected. By stronglyinhibiting the adherence, the adhesion of the pathogens to theepithelial host cells can be prevented so that by the usual defensemechanisms, such as ciliary movement of the epithelial cells, mucussecretion, mass flow of body fluids and the like, the pathogens can beeliminated.

Finally, the pharmaceutical compositions of the invention can be usedfor preventing or fighting Neisseria infections. Preferably, forpreventive applications the proteins of the invention or fragmentsthereof are used for the production of a vaccine for activeimmunization, or antibodies of the invention are used for the productionof a passive vaccine applicable as a therapeutic. The applicationsdescribed above also apply to the pharmaceutical compositions anddiagnostic compositions described below.

The subject matter of the invention further relates to nucleic acidmolecules encoding a protein or a biologically active fragment thereoffrom bacteria of the genus Neisseria having the amino acid sequencedescribed in Seq ID No. 3. Such nucleic acid molecules preferably havethe nucleotide sequence described in Seq ID No. 3, in particular the oneof the described coding region. The invention also relates to nucleicacid molecules the sequence of which deviates from the sequences of themolecules mentioned above due to the degeneration of the genetic code.Also nucleic acid molecules are the subject matter of the invention thathybridize to the nucleic acid molecules mentioned above (for thedefinition of the term “hybridization” see above). For the possiblevariants of the nucleic acid molecules the same is true what has alreadybeen described in connection with the nucleic acid molecules encodingOrfA.

The invention also relates to, vectors containing the described nucleicacid molecules, in particular those in which they are linked toregulatory DNA elements for the expression in prokaryotic or eukaryoticcells, as well as to host cells that contain such vectors or that aregenetically manipulated with the described nucleic acid molecules.

The invention also relates to proteins encoded by the nucleic acidmolecules described above and to proteins containing amino acidsequences that immunologically cross-react with the amino acid sequencedepicted in Seq ID No. 3 or fragments thereof. In the context of thisinvention they are called OrfI proteins. The protein from Neisseriagonorrhoeae having the amino acid sequence depicted in Seq ID No. 3shows in the T7 expression system an apparent molecular weight of about18 kd. A homology to presently known proteins could not be shown.Experimental data indicate that the protein is located intracellularlyand possibly has a regulatory function.

This protein can be produced by a method in which a host cell describedabove is cultivated under conditions allowing the expression of theprotein and in which the protein is obtained from the cells and/or theculture supernatant. Therefore, the invention also relates to proteinsobtainable by such a method.

The invention also relates to antibodies against a protein describedabove or a fragment thereof as well as to nucleic acid molecules havinga length of at least 12 nucleotides and specifically hybridizing to anucleic acid molecule described above. Preferably, the molecules have alength of more than 15 nucleotides and particularly preferably of morethan 20 nucleotides.

The invention further relates to pharmaceutical compositions containinga nucleic acid molecule, protein, biologically active fragment thereofand/or an antibody described above and, optionally, a pharmaceuticallyacceptable carrier.

The invention further relates to diagnostic compositions containing thenucleic acid molecules, proteins, biologically active fragments thereofand/or antibodies described above.

The subject matter of the invention further relates to nucleic acidmolecules encoding a protein or a biologically active fragment thereoffrom bacteria of the genus Neisseria that has the amino acid sequencedepicted in Seq ID No. 4. Such nucleic acid molecules preferably havethe nucleotide sequence depicted in Seq ID No. 4, in particular the oneof the indicated coding region. The invention also relates to nucleicacid molecules the sequences of which deviate from the nucleotidesequence of the above-mentioned molecules due to the degeneration of thegenetic code. Furthermore, the subject matter of the invention alsorelates to nucleic acid molecules hybridizing to the above-mentionednucleic acid molecules (for the definition of the term “hybridization”see above). The same applies to possible variants of the nucleic acidmolecules as has already been described in connection with the nucleicacid molecules encoding OrfA.

In a preferred embodiment the above-described nucleic acid moleculesencode a protein that is able to form a complex with the protein PilCand therefore shows an ability of adherence to human cells.

The invention also relates to vectors containing the described nucleicacid molecules, in particular those in which they are linked toregulatory DNA elements for the expression in prokaryotic or eukaryoticcells, as well as to host cells that contain such vectors or that havebeen genetically manipulated with the above-described nucleic acidmolecules.

The invention also relates to proteins encoded by the above-describednucleic acid molecules and to proteins containing the amino acidsequences that immunologically cross-react with the amino acid sequencedepicted in Seq ID No. 4 or parts thereof. These are called OrfB in thecontext of the present invention. The protein from Neisseria gonorrhoeaehaving the amino acid sequence depicted in Seq ID No. 4 shows in the T7expression system an apparent molecular weight of about 57 kd. Ahomology to presently known proteins could not be shown. Experimentaldata indicate that the protein is, like OrfA, located at the cellsurface and is accessible from the outside. Furthermore, it obviouslyalso possesses the ability to form a complex with the protein PilC andto induce either alone or in combination with OrfA the adhesion to humancells.

This protein can be produced by a method in which an above-describedhost cell is cultivated under conditions allowing the expression of theprotein and in which the protein is obtained from the cells and/or theculture supernatant. Therefore, the invention also relates to proteinsobtainable by such a method.

The invention also relates to antibodies against an above-describedprotein or fragment thereof, as well as to nucleic acid molecules havinga length of at least 12 nucleotides and specifically hybridizing to anabove-described nucleic acid molecule. Preferably, such molecules have alength of more than 15 nucleotides and particularly preferred of morethan 20 nucleotides.

Furthermore, the invention relates to pharmaceutical compositionscontaining an above-described nucleic acid molecule, protein,biologically active fragment thereof and/or antibody and, optionally,pharmaceutically acceptable carriers.

The subject matter of the invention further relates to diagnosticcompositions containing the above-described nucleic acid molecules,proteins, fragments thereof and/or antibodies.

Illustration of the figures and the sequence protocols:

FIG. 1 schematically shows the construction of the plasmid pES25.

FIG. 2 shows the nucleotide sequence (SEQ ID No. 1) of the orf generegion, starting from position 1 at the modified BglI cleavage site andending with position 3260, the last nucleotide of the HindIII cleavagesite. Restriction cleavage sites, ribosome binding sites (Shine-Dalgarnosequences) and promoter sequences (−35 and −10 regions) are labeled.

SEQ ID No. 1 further shows the amino acid sequences of the proteinsOrfI, OrfA and OrfB encoded by the orf gene region. The amino acids ofthe lipoprotein signal sequence of OrfA are written in italic, thecleavage sites of the lipoprotein signal peptidase II is labeled withthe tip of an arrow. The amino acid cysteine that represents the aminoterminal of the processed OrfA lipoprotein and is modified to glycerylcysteine with fatty acid is marked with a circle. The first seven aminoacids of OrfB that are similar to a typeIV-pilin-signal sequence arewritten in bold. The labeling between amino acids 7 and 8 and between 11and 12 characterize potential cleavage sites analogous to the processingof the typeIV-pilin.

Seq ID No. 2 shows the nucleotide sequence of the gene region encodingOrfA as well as flanking sequences. The amino acid sequence of OrfA isdepicted, too.

Seq ID No. 3 shows the nucleotide sequence of the gene region encodingOrfI as well as flanking sequences. The amino acid sequence of OrfI isdepicted, too.

Seq ID No. 4 shows the nucleotide sequence of the gene region encodingOrfB as well as flanking sequences. The amino acid sequence of OrfB isdepicted, too.

The examples illustrate the invention.

EXAMPLES Example 1 Method for the Isolation of the Lipoprotein AdhesinOrfA

During the chromatographic purification of the PilC protein a decisiveobservation with regard to the identification of the new adhesin ofNeisseria gonorrhoeae of the invention was made. A recombinant PilCprotein was used that was amplified by an oligo-histidine region withsix histidine residues (His₆-tag) in order to make the chromatographicpurification easier (Rudel et al., Nature 373, 357-359, 1995). Theamplification of the protein by the histidine hexapeptide makes theselective binding to a nickel-nitrilotriacetate-agarose matrix (Ni-NTAmatrix) possible. After the cell wall fraction produced from cultures ofa pilus-free PilC overexpression strain N560 (Rudel et al., see above)from Neisseria gonorrhoeae had been extracted, the extract was loaded onan Ni-NTA chromatography matrix. Usually, for the method that wasdeveloped for the purification of recombinant PilC unspecifically boundmaterial was removed by extensive washing with a buffer containingimidazole. However, in the first elution fraction a protein of 36 kd(OrfA) could be identified together with PilC in an approximatelyequimolar ratio.

For the preparation of the PilC-OrfA protein fraction the strain N560from Neisseria gonorrhoeae was plated on 30 GC-agar plates and incubatedin 5% CO₂ at 37° C. for 20 hours. The GC-agar medium (GC agar base,Becton Dickinson, Heidelberg) contained the usual additional factorsnecessary for the growth of Neisseria gonorrhoeae (0.1 mg vitamin B12,10 mg adenine, 0.3 mg guanine, 100 mg glutamine, 1 mg cocarboxylase, 0.3mg thiamine, 259 mg L-cysteine, 11 mg L-cystine, 1.5 mg arginine, 5 mguracil, 0.2 mg Fe(NO₃)₃, 2.5 mg nicotineamide-adenine dinucleotide, 0.13mg p-aminobenzoic acid and 1 g dextrose per 1 liter of medium) that wereadded as a sterile filtrate to the GC basis medium after heatsterilization. Furthermore, the so supplemented GC agar medium contained5 μg/ml tetracycline and 100 μM IPTG. The bacterial lawns were removedwith cotton pads, transferred to 30 ml of washing buffer (Tris-HCl pH8.0 with 0.15 M NaCl) and centrifuged at 4,000 rpm, 4° C. for 15 minutes(Du Pont Sorvall Centrifuge RC-5B, Rotor SS-34). The cell sediment wasagain resuspended in 30 ml of washing buffer, and the bacteria werebroken up by ultrasonic homogenization after lysozyme and 5 mM EDTA Na₂had been added. Intact bacteria were separated by centrifugation at5,000 rpm at 4° C. for 15 minutes. The cell coats of the lysed bacteriawere sedimented by centrifugation of the supernatant at 20,000 rpm at 4°C. for 60 minutes and taken up in 10 ml of washing buffer additionallycontaining 10% glycerine, 10 mM MgCl₂ and 2% Triton X-100. After anincubation of 45 minutes at 37° C. they were centrifuged again (20,000rpm, 4° C. for 60 minutes) and the membrane sediment suspended in 10 mlof washing buffer with 10% glycerine, 10 mM MgCl₂ and 2% LDAO(N,N-Dimethyldodecylamin-N-oxide) and incubated at 37° C. for 60minutes. After they were centrifuged again (20,000 rpm, 4° C. for 60minutes), the supernatant containing the biologically active PilC-OrfAcomplex was subjected to a nickel-chelate-affinity chromatography forfurther purification. For this purpose a Ni-NTA-gel matrix (300 ml bedvolume) was washed with 5 bed volumes of aqua bidest. and loaded with 10ml of the supernatant. Unspecifically bound proteins were removed byelution with 5 column volumes of 50 mM imidazole in PBS buffer pH 8.0.After the column had been washed again with 5 to 10 bed volumes 20 mMsodium phosphate pH 7.5 with 0.15 M NaCl (PBS buffer) the biologicallyactive PilC-OrfA complex was eluted with a citrate/phosphate buffer (10mM citric acid, 1 M sodium phosphate, pH 3.5, 10% glycerin, 0.15 M NaCl)in the first elution fraction and instantly neutralized with a 1 MNa₂HPO₄ solution. The eluate containing PilC and OrfA was frozen inliquid nitrogen and strored at −70° C.

Example 2 Isolation of the Polynucleotide Sequence Carrying the Orf-GeneRegion

To further characterize the 36 kd OrfA protein, mice were immunized withthe PilC-36 kd protein fraction. The 36 kd protein proved to be veryimmunogenic. With the antibodies obtained this way a pBA plasmid genelibrary of the Neisseria gonorrhoeae MS11 genome in E. coli GC1 wasscreened for the presence of antigens. Several clones showing a positivereaction were isolated and clone H1967 was chosen for furthercharacterization.

The library plasmid pES25 (FIG. 1) of clone H1967 contained a genomicfragment of approximately 11 kb, cloned in vector pBA. Restrictionfragments of the total region were subcloned in pUC and pBluescript KS(+) vectors, respectively. On the basis of the expression of the derivedplasmids in minicells and immunoblotting analyses subclones were chosenproducing the 36 kd protein. The subclones were used for sequencing. Thesequences were determined by directly sequencing restriction fragments,by sequencing continuously shortened ExoIII nuclease fragments of theBglI-PstI fragment (positions 1 to 2560 of Seq ID No. 1), as well as bysequencing PCR amplified fragments.

The region depicted in SEQ ID No. 1 starting from the BglI cleavage site(position 1) to the HindIII cleavage site (position 3260) had three openreading frames with a high coding probability with each reading framebeginning with the start codon ATG, having a ribosome binding site thatprecedes the start codon in a suitable distance (S.D. sequence) andending with a stop codon.

The three reading frames have the same orientation. The first openreading frame starts at position 136 of the sequence depicted in SEQ IDNo. 1 and ends at position 450 with the stop codon TAA. The encodedprotein was called OrfI and had an apparent molecular weight of 18 kd inthe T7 expression system.

No significant homologues could be identified by sequence comparison inthe EMBL gene library (Release 43.0 from 6/95) and in the SwissProt databank (Release 31.0 from 3/95), neither on a nucleotide sequence levelnor on an amino acid sequence level.

The second open reading frame starts at position 583 and ends atposition 1545 with the stop codon TGA. It encodes the OrfA proteinhaving an apparent molecular weight of 36 kd in the T7 expressionsystem. Also to this sequence no significant homologues could bedetected via data base search. The sequence analysis by means of theprotein analysis program “Motifs” (GCG Genetics Computer Group, Inc.,Madison, Wis., USA) showed, however, a complete homology of theN-terminus of OrfA to lipoprotein specific signal sequences (position583 to 636). The characterization of OrfA as a lipoprotein could besubstantiated by experiments (vide infra).

The third open reading frame starts at position 1585 and ends atposition 3114 with the stop codon TGA. The protein OrfB hereby encodedhas an apparent molecular weight of 57 kd in the T7 expression system.Also to this reading frame no homologue could be identified via database search.

As a structural peculiarity the amino terminus of the OrfB sequencedisplays a signal sequence showing similarities to the type IV-prepilinsignal sequence. At positions 8 and 12 of the amino acid sequence thereis phenylalanine so that there are in addition two possible cleavagesites for the type IV pilin signal peptidase. It can be derived herefromthat OrfB presumably is a secreted protein.

The molecular weights of all the three gene products measured in the T7expression system correspond to the values theoretically calculated fromthe sequence. The separation of the expression products by means of gelelectrophoresis showed that the OrfB-band was significantly weaker thanthe OrfA-band in all the cases. This points to a weaker expression ofOrfB.

Two regions showing a sequence homology to the promoter regions wereidentified. One of them is located in front of the orfl gene, the secondone in front of the orfA gene, each leaving an appropriate distance (SEQID No. 1). Therefore, it can be assumed that orfA and orfB form atranscription unity.

The analysis of the Neisseria gonorrhoeae MS11 genome after ClaI andMluI digestion showed a complex band pattern in Southern hybridizationwith plasmid pES-8 as sample. This fact indicates the existence ofseveral copies of the orf-gene region, probably of three copies, in thegenome of Neisseria gonorrhoeae MS11. If all these loci are expressed,if they are subjected to antigenic variations like, for example, theNeisseria genes pilS and opa, and if the flanking regions of the orfgene region are involved in the sequence repetitions, is presently notknown.

Example 3 Characterization of the Localization of OrfA and OrfB on theCell Surface

In order to experimentally prove the lipoprotein nature of orfAderivable from the perfect structure homology of the amino terminus oforfA to lipoprotein signal sequences, both N. gonorrhoeae and E. colirecombinants transformed with the orf-gene region were labeled with [³H]palmitate. The results of the labeling show that in all the cases, bothwith N. gonorrhoeae and with the E. coli recombinants, lipoproteins inthe corresponding molecular weight range could be identified. While withN. gonorrhoeae several proteins were labeled and the labeled band couldnot be precisely assigned since there was no orfA⁻ mutant available, theorfA recombinants of E. coli showed in comparison to the control strainunambiguously only one additional band having the molecular weight ofOrfA. An OrfA fusion protein that was tested in addition and wasamplified at the carboxy-terminal by a fusion of 3 kd, also had a [³H]palmitate labeling and migrated to a position precisely corresponding tothe molecular weight that was, as expected, increased due to the fusion.

When prepared cell coats were treated with detergents, OrfA showed asolubility that is typical of proteins of the outer membrane. Byseparating the cell coat by means of density-gradient centrifugation itcould be confirmed by means of marker proteins that OrfA was located inthe outer membrane of N. gonorrhoeae. Also with orf recombinants of E.coli, OrfA was shown to be a protein component of the outer membrane bymeans of said method.

The accessibility of the cell surface was proven by means of animmunofluorescence test both for OrfA and OrfB. A defective pilC mutantof Neisseria gonorrhoeae the two pilC genes of which were switched offis labeled by the PilC-OrfA antiserum in the same way as recombinant E.coli strains carrying the orf-gene region. The non-transformed controlstrain showed, as was to be expected, a negative reaction. A positivereaction in the immunofluorescence test of N. gonorrhoeae and orfrecombinant E. coli strains could be brought about by means of OrfA andOrfB specific antisera using purified fusion proteins of either OrfA orOrfB. for the production of these antisera. If antisera were useddirected against an OrfI fusion protein, the immunofluorescence testwith N. gonorrhoeae was negative. From this it can be deduced that OrfAand OrfB are located on the cell surface and are accessible from theoutside, whereas OrfI probably is located intracellularly.

The surface localization of OrfA and OrfB could only be proven inrecombinant E. coli strains carrying the whole orf region.

Example 4 Adhesin Property of the OrfA-PilC Complex

As mentioned above OrfA could be obtained in pure form by chromatographyon an Ni-NTA-chelate matrix due to its affinity to PilC. Since thefunction of PilC as pilus associated adhesin had been proven and thedirect binding of PilC to human ME-180 cells had been known, it wasobvious to test the adherence property of the PilC-OrfA complex. Theexperiments were performed with the E. coli strain HB101 (E141) since itdoes not possess the mannose specific typeI pili and shows almost nobinding to human ME-180 and Chang epithelial cells. After thetransformation of HB101 with the plasmid pES25, no adherence, neither toME-180 nor to Chang cells, could be mediated. If the same recombinants,however, were pre-incubated with PilC protein, a strong adherence toChang epithelial cells but not to ME-180 cells could be induced (TableI). TABLE I OrfA-dependent modulation of the PiIC mediated adhesinfunction Adherence to human epithelial cells ME180 cells Chang cells N.gonorrhoeae, Orf+ PilCi+, Pili+ +++ + N. gonorrhoeae, Orf+, PilC+,Pili− + +++ E. coli (E141) − − E. coli (E141) + PilC (extern) − − E.coil (H2561) − − E. coli (H2561) + PilC (extern) − − E. coli (H2560) − +E. coli (H2560) + PilC (extern) − +++

Three independent experiments were evaluated, whereby the adherence ofNeisseria was determined using 500 cells and the adherence of the E.coli strains was determined per epithelial cell.

+++100%, ++50%, +25% adherence.

E. coli E141=E. coli strain HB101 without plasmid; E. coli H2561=E. colistrain HB101 with plasmid pBA; E. coli H2560=E. coli strain HB101 withplasmid pES25 The plasmid pES25 (FIG. 1) is a pBA vector containing agenomic fragment from Neisseria gonorrhoeae of approximately 11 kbcarrying the coding regions orfA, orfB and orfI.

The E. coli strain H2560 was deposited at the Deutsche Sammiung furMikroorganismen (DSM, Braunschweig, Germany) under the DSM-AccessionNumber DSM 10257.

The result obtained is surprising since pilus carrying Neisseria bind toME-180 cells with a significantly higher affinity than to Changepithelial cells. This result can be put down to the fact that PilC hasdifferent adherence properties depending on its localization. As anadhesin component in the pilus PilC preferably binds to receptors of theME-1800 cell surface, whereas as an adhesin located on the cell surfacein the complex with OrfA PilC preferably recognizes receptors on Changepithelial cells. If in the latter case adhesin properties also can beascribed to OrfA and/or OrfB, is presently not known.

The results obtained for recombinant E. coli strains could be reproducedwith the same result with N. gonorrhoea. If the pilus-free strain N 300(P-Opa-), which hardly binds to ME-180 or Chang cells, is pre-incubatedwith purified PilC, the adherence to Chang epithelial cells can besignificantly increased.

The described experimental approaches obviously provide for a model thatis suitable to analyze a mechanism for the modulation of the adherenceproperties, how they can in cascade-like order effect the increasinglystrong adherence of the pathogens to the host cells or how they can bethe basis for the tissue tropism.

1. An isolated nucleic acid molecule encoding a lipoprotein or abiologically active fragment of said lipoprotein that mediates adhesionof Neisseria cells to human cells from a bacteria of the genusNeisseria, selected from the group consisting of (a) a nucleic acidmolecule comprising a nucleotide sequence encoding a protein comprisingSEQ ID NO: 4; (b) a nucleic acid molecule comprising a nucleotidesequence having 95% sequence identity to a nucleotide sequence encodinga protein comprising SEQ ID NO:4 due to the degeneracy of the geneticcode; (c) a nucleic acid molecule comprising a nucleotide sequence thathybridizes under stringent hybridization conditions of 0.2×SSC, 0.1% SDSand 68° C. to (i) the complement of a nucleotide sequence encoding aprotein comprising SEQ ID NO:4, (ii) the complement of a nucleotidesequence which is 95% identical to a nucleotide sequence encoding aprotein comprising SEQ ID NO:4.
 2. The nucleic acid molecule accordingto claim 1, wherein the nucleic acid molecule originates from apathogenic Neisseria species.
 3. The nucleic acid molecule according toclaim 2, wherein the Neisseria species is Neisseria gonorrhoeae orNeisseria meningitidis.
 4. The nucleic acid molecule according to claim1, wherein the lipoprotein or biologically active fragment of saidlipoprotein has the ability to adhere to human cells.
 5. A vectorcomprising the nucleic acid molecule according to claim
 1. 6. The vectoraccording to claim 5, wherein the nucleic acid molecule is operativelylinked to at least one regulatory DNA element allowing the expression ofsaid nucleic acid molecule in a prokaryotic or an eukaryotic cell.
 7. Ahost cell comprising a vector according to claim
 5. 8. A host cellcomprising the nucleic acid molecule according to claim
 1. 9. Anisolated nucleic acid molecule having a length of at least 12nucleotides specifically hybridizing under stringent hybridizationconditions of 0.2×SSC, 0.1% SDS and 68° C. to a nucleic acid moleculeaccording to claim 1.